Hypothesis

Urolithin A (UroA) does more than initiate mitophagy; it also potentiates lysosomal acidification by upregulating the V‑ATPase proton pump, thereby sealing the mitophagic flux. This coupling ensures that damaged mitochondria are not only sequestered but efficiently degraded, linking microbial metabolism to organelle quality control in the gut epithelium.

Mechanistic Rationale

• UroA activates SIRT1, which deacetylates and activates TFEB, a master lysosomal biogenesis regulator [1].
• TFEB drives transcription of V‑ATPase subunits (ATP6V0A1, ATP6V1B2) and lysosomal hydrolases, increasing proton pumping capacity.
• Concurrently, UroA‑induced AMPK activation phosphorylates the V‑ATPase assembly factor RASSF1A, stabilizing the holoenzyme on lysosomal membranes (novel link).
• Enhanced lysosomal acidity accelerates cathepsin activity, completing mitophagy that was initiated via PINK1/Parkin or alternative pathways [2,3].
• Intestinal epithelial cells, the primary site of UroA production, thus experience a feed‑forward loop: higher local UroA → stronger lysosomal acidification → more efficient mitophagy → reduced ROS and barrier preservation [4].
• Failure to acidify lysosomes leads to accumulation of autophagosomes, a hallmark of incomplete mitophagy observed in aging mucosa.

Testable Predictions

1. In human intestinal organoids, UroA treatment will raise lysosomal pH-sensitive dye signal (indicating lower pH) and increase V‑ATPase subunit mRNA, effects blocked by SIRT1 inhibitor EX527.
2. Organoids treated with UroA will show increased colocalization of mito‑Keima (mitophagy reporter) with LAMP1, and this will be abolished by V‑ATPase inhibitor bafilomycin A1.
3. Microbiome‑defined mice lacking ellagitannin‑transforming bacteria will not exhibit lysosomal acidification or mitophagy flux increase after dietary ellagitannin supplementation, whereas colonization with a engineered Gordonibacter strain producing UroA will restore both.
4. Circulating monocytes from older adults will show a positive correlation between fecal UroA levels, lysosomal acidity (measured by LysoSensor), and mitophagy flux (mito‑Keima), independent of systemic inflammation.

Experimental Approach

• In vitro: Human colono‑derived organoids ± UroA (5 µM) ± EX527 (10 µM) or BafA1 (100 nM). Measure lysosomal pH (LysoSensor Blue/Red ratio), V‑ATPase ATPase activity, TFEB nuclear translocation (immunofluorescence), and mito‑Keima flux.
• In vivo: Germ‑free mice colonized with either a defined microbiota lacking urolithin‑producing taxa or the same community spiked with a UroA‑secreting Gordonibacter mutant. Feed ellagitannin‑rich diet (0.5% punicalagin) for 8 weeks. Assess intestinal epithelial lysosomal pH, mitophagy (mito‑Keima TEM), barrier integrity (FITC‑dextran permeability), and systemic inflammaging markers (IL‑6, TNFα).
• Human pilot: Collect stool and blood from 60 volunteers aged 60‑80. Quantify fecal UroA (LC‑MS), lysosomal acidity in isolated monocytes (LysoSensor), and mitophagy flux (mito‑Keima flow cytometry). Model relationships with linear regression, adjusting for age, BMI, and CRP.

If UroA’s effect on lysosomal acidification is required for complete mitophagy, inhibiting either SIRT1 or V‑ATPase will uncouple mitophagy initiation from completion, leading to autophagosome accumulation despite UroA presence. This hypothesis directly links a microbial metabolite to lysosomal physiology, offering a clear, falsifiable mechanism that extends beyond initiating mitophagy to ensuring its finish.